Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization

Abstract

Rsp5 ubiquitin ligase belongs to the Nedd4 family proteins, which affect a wide variety of processes in the cell. [ ] Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for a tpk1tpk3 mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA Rsp5 protein using an anti-PKA-phosphopeptide antibody that Rsp5 is phosphorylated in PKA sites. [ ] Rsp5 contains the sequence 758-RRFTIE-763 in the catalytic domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28oC, and did not grow at 37oC and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A causes mutant Rsp5 to locate in internal spots. Rsp5-T761A protein is phosphorylated or not depending on the growth conditions. Thus, Rsp5 is phosphorylated by PKA in T761 and other site(s) and mimicking phosphorylation in T761 affects its localization and function.